The origin of local and global stability and dynamics of proteins and protein complexes have long been of interest and are now of particular importance in the emerging field of protein design and engineering. One approach to the determination of molecular structure and dynamics in proteins involves the use of high pressure NMR spectroscopy. The use of pressure to probe the global and local stabilities of proteins and protein complexes apparently offers the opportunity to selectively destabilize certain classes of interactions with respect to others based on significant differences in the standard free energies and volume changes associated with their disruption. For example, protein complexes that are stabilized by intermolecular salt links are often exquisitely sensitive to pressure and dissociate at pressures of a few hundred bar (Silva and Weber, 1993, Annu. Rev. Phys. Chem. 44:489-113; Weber and Drickamer, 1983, Quarterly Rev. Biophys. 1689-112; Robinson and Sligar, 1995, Meth. Enz. 259:395-427).
Since the pioneering work of Benedek and Purcell (1954, J. Chem. Phys. 22:2003-2012) two types of approaches to achieving high pressure NMR capability have evolved. The high pressure probe technique takes the design principle that the entire RF coil and sample are to be pressurized. This original design strategy has been adopted and extended by Jonas and coworkers (Jonas and Jonas, 1994, Annu. Rev. Biophys. Biomol. Struct., 23:287-318; Jonas et al., 1993, Magn. Reson., Series B, 102:299-309). The second general approach is the high pressure cell technique which employs thick walled glass (Wagner, 1980, FEBS Lett., 112:280-284), Vespel (Vanni et al., 1978, J. Magn. Reson., 29:11-19) or sapphire (Roe, 1985, J. Magn. Reson., 63:388-391) tubes of various construction or reinforced quartz capillaries (Yamada, 1974, Rev. Sci. Instrum., 45:640-642) which fit directly into a standard NMR probehead.
Previously, others have disclosed pressure cells for use in NMR measurements. U.S. Pat. No. 5,045,793 (Rathke) describes the design and use of a high pressure probe with emphasis on the RF coil design. The whole probe was pressurized in order to make the measurements. An operating pressure of 4750 psi (327.5 bar) for 12 hours was used. U.S. Pat. No. 5,122,745 (Smith et al.) describes a method and apparatus for determining molecular dynamics of material. The apparatus entails a high pressure cell where pressures of up to 50 atmospheres (50.7 bar) were achieved. The high pressure cell is combined with an NMR probe to analyze samples. Other reports in the literature, where high pressure was approached, illustrate use of only simple homonuclear NMR spectroscopy at pressure above 1 kilobar (see Jonas and Jonas 1994, Ann Rev. Biophys. Biomol. Struct. 23:287-318).
A sapphire tube was used by Roe (1985, J. Magn. Reson., 63:388-391) to achieve high pressures. The sample tube was mounted to a titanium alloy flange with a single component epoxy adhesive. Pressures of 5000 psi (345 bar) were routinely used. The sapphire tube burst at a pressure of 14,500 psi (1 kilobar).
Thus while it is of great interest to apply techniques such as NMR spectroscopy to the analysis of pressure-induced transitions in protein complexes, it has not been possible heretofore to subject samples to kilobar pressures. What is needed, therefore, is a pressure cell that can withstand high pressures while allowing state-of-the-art NMR measurements.